Isolated aquaporin in its closed conformation

ABSTRACT

The invention relates to an isolated aquaporin having a bound ligand, wherein said ligand close the conformation of said aquaporin and inhibit and/or reduce water transport of said aquaporin, and/or a high resolution structure of an isolated aquaporin in a closed conformation characterised by the coordinates deposited at the Protein Data Bank ID:1Z98, a crystal of said isolated aquaporin as well as the coordinates defining said crystal and the use of said aquaporin, and the use of the high-resolution structure as defined by the coordinates deposited at PDB ID:1Z98, and a method to produce said aquaporin.

This application is a Divisional of U.S. Ser. No. 12/066,152, filed 15May 2009, which is a National Stage Application of PCT/SE2006/001036,filed 8 Sep. 2006, which claims benefit of Serial No. 0501999-7, filed 9Sep. 2005 in Sweden and which applications are incorporated herein byreference. To the extent appropriate, a claim of priority is made toeach of the above disclosed applications.

FIELD OF INVENTION

The invention relates to an isolated aquaporin having a bound ligand,wherein said ligand closes the conformation of said aquaporin andinhibit and/or reduce water transport of said aquaporin, and/or a highresolution structure of an isolated aquaporin in a closed conformationcharacterised by the coordinates set forth in Appendix 1, a crystal ofsaid isolated aquaporin as well as the coordinates defining said crystaland the use of said aquaporin, the use of the high-resolution structureas defined by the coordinates set forth in Appendix 1, and a method toproduce said aquaporin.

BACKGROUND OF INVENTION

Water is the medium of life. Since biological membranes have onlylimited intrinsic water permeability cells maintain the flux of waterinto and out of the cell via a family of water-specific, membraneprotein channels called aquaporins (1). Members of the aquaporin familyare found in archea, eubacteria and eukaryotes, including fungi, animalsand plants. They serve an astonishing variety of physiological functions(5-7) and are easily identified by sequence similarity across allkingdoms of life. In higher eukaryotes, water transport activity ofaquaporins is frequently regulated by phosphorylation, pH and osmolarity(6-8). Aquaporins in plants and animals are highly conserved and formlarge protein families with 35 members in higher plants (9) and 13members in humans (5,10).

Based upon phylogenetic analyses, plant aquaporins are further dividedinto four subfamilies and their presence in primitive plants such as thebryophyte Physcomitrella patens implies that this specialization wasalready present in an ancient plant-ancestor (11). There are 13remarkably conserved plasma membrane aquaporins (Plasma membraneIntrinsic Proteins or PIPs) which are all regulated, and these furtherseparate into two distinct phylogenetic groups (PIP1 and PIP2).

Closure of the plant aquaporin SoPIP2;1 of spinach (formerly calledPM28A(2)) has been reported to be triggered by the dephosphorylation oftwo serine residues: Ser115 in the cytosolic loop B (conserved as Ser in12, and as Thr in 1, of the 13 Arabidopsis PIPs) and Ser274 in theC-terminal region (2,3) (conserved as Ser in 7, and as Thr in 1, of the8 Arabidopsis PIP2s). Both residues are situated in consensusphosphorylation sites. Furthermore, the simultaneous closure of allArabidopsis PIPs upon anoxia was recently reported to depend upon theprotonation of a strictly conserved histidine residue in loop D (4),which corresponds to His193 in SoPIP2;1 (SEQ ID NO: 33). It is anintriguing observation that distinct chemical signals acting on residueswell separated in sequence induces an identical physiological responsewithin PIPs. While a number of structures have been reported for water(12-16) and glycerol (17) channels, no plant aquaporin structure has yetbeen determined at high resolution. Gonen et al. (15) reports alow-resolution structure of AQP0. At this resolution (3 Å) watermolecules cannot be seen and the authors are not able to conclude thatthe structure represents a closed aquaporin. This is also clearly statedby the authors in the article (p 194-195: “We note, however, that ourresolution is currently limited to 3 Å, and even if a pore appears to bein a closed conformation, it might still be permeable to solutes.”).Furthermore, a high-resolution structure of AQP0 (16) with an openconformation show no global change in the structure as compared to thelow-resolution AQP0 structure reported in ref 15. Thus, it is likelythat the structure in ref 15 represents an open aquaporin. Recently anadditional report (35) arrives at the same conclusion that the structureof AQP0 reported in ref 15, as well as in ref 16, is open and not closedto water transport.

In addition, the low-resolution structure of AQP0 presented in Gonen etal. (15) is based on a proteolytically cleaved AQP0. Thus, both the N-and C-terminal regions of the protein are cleaved off and can thereforenot participate in closing the pore (36). Kukulski et al. (37) depicts a5 Å low-resolution structure of an aquaporin that in a previouspublication by the same authors had been shown to be open (28). However,from a 5 Å low-resolution structure it is impossible to see if the poreis open or closed.

Furthermore, no gating mechanisms have been unambiguously demonstrated.Therefore it is crucial to establish the atomic structure of anaquaporin in its closed formation. Structural information of the closedconformation is necessary for understanding the mechanism of gating andfor structure-based design and development of organic compounds,peptides or antibodies that either stabilize the open conformation orthe closed conformation. By obtaining the structure of a closedaquaporin it will for the first time be possible to use that particularstructure to modify the gating. This can in plants be done by directgenetic engineering of aquaporins in order to improve stress tolerance,e.g. against drought stress. In mammalian species pharmaceuticalcompounds that stabilize the closed or the open conformation ofaquaporins can be designed based on the closed conformation of theaquaporin SoPIP2;1 (SEQ ID NO: 33) from the plasma membrane of the plantspinach. Such inhibitors and activators are candidate pharmaceutical andcosmeceutical compounds, e.g. antiperspirants. Aquaporins are alsoimportant for cell migration during angiogenesis, wound healing, tumourspread and organ regeneration (27), processes that therefore can bemodulated by pharmaceutical compounds interacting and modifying thegating of aquaporins. Dysfunction of human aquaporins is associated withclinically important diseases such as polyuria in kidney diseases.Conversely, increased water retention is associated with congestiveheart failure, liver cirrhosis and nephritic syndrome. Also pathologicalskin conditions such as anhidrosis, hyperhidrosis and conditions wherethe transepidermal water loss is deviating from normal conditions couldbe targets for aquaporin inhibitors and activators. Moreover, brainedema, glaucoma and skin burns could be treated by inhibitors andactivators of aquaporins. The cosmeceutical applications of aquaporininhibitors and activators include not only antiperspirant but alsodermatological conditions where one wants to influence thetransepidermal water loss. The atomic structure of the closedconformation of SoPIP2;1 (SEQ ID NO: 33) can also be used for designingnovel in silico and in vitro screening systems for pharmaceuticals andcosmeceuticals acting as modulators of aquaporin gating and function.Knowledge of the atomic structure of the closed conformation can also beused to design, and also to screen for, peptides and antibodies thatinteract with certain epitopes on aquaporins and thus effect activityand gating.

SUMMARY OF THE INVENTION

The object of the present invention is to solve the above, discussedproblems in connection with aquaporins and the gating mechanism ofaquaporins. This object is achieved by the present invention asspecified below.

The object of the present invention, is the isolation and determinationof the structure of an aquaporin and thereby enable the possibility tosolve all the above mentioned problems.

The invention relates in one aspect to an isolated aquaporin having abound ligand, wherein said ligand closes the conformation of saidaquaporin and inhibit and/or reduce water transport of said aquaporin,and/or a high resolution structure of an isolated aquaporin in a closedconformation characterised by the coordinates set forth in Appendix 1, acrystal of said isolated aquaporin as well as the coordinates definingsaid crystal and the use of said aquaporin, the use of thehigh-resolution structure as defined by the coordinates set forth inAppendix 1, and a method to produce said aquaporin.

In a second aspect, the invention relates to a crystal of an isolatedaquaporin in its closed conformation having an atomic structurecharacterised by the coordinates set forth in Appendix 1.

In a third aspect the invention relates to a method of producing anisolated aquaporin having a closed conformation comprising the steps;providing said aquaporin, adding a ligand to said aquaporin; producingcrystals and obtaining an aquaporin having a ligand bound to saidaquaporin, wherein said ligand closes the conformation of said aquaporinand inhibit and/or reduce water transport through said aquaporin.

In a fourth aspect the invention relates to the use of said isolatedaquaporins or said crystal of said aquaporin as well as the coordinatescharacterising said crystal.

In a final aspect the invention relates to the use of a ligand bindingto the cytoplasmic side to close the conformation of an aquaporin.

Further advantages and objects with the present invention will bedescribed in more detail, inter alia with reference to the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows sequence comparisons between selected aquaporins. Sequencecomparisons between selected aquaporins from plants, animals andbacteria. SoPIP2;1 (SEQ ID NO: 33) is indicated with an arrow andimportant conserved residues among PIPs or PIP2s are indicated at thetop together with the number of the corresponding residue in SoPIP2;1(SEQ ID NO: 33). At, Arabidopsis thaliana, SEQ ID NO: 9, see also SEQ IDNOs: 10-13, 23-30, 53-55, 58-60, 64, 65, 68, 73-77, 81-85, 87, 88, and91; Zm, Zea mays, SEQ ID NO: 3, see also SEQ ID NOs: 4-8, 16-22, 56, 57,61-63, 66, 67, 69-72, 78-80, 86, 89, and 90; Pa, Picea abies, SEQ ID NO:14, see also SEQ ID NO: 31; Pp, Physcomitrella patens, SEQ ID NOs: 15,see also SEQ ID NO: 32; So, Spinacea oleracae, SEQ ID NO: 33; Hs, Homosapiens, SEQ ID NO: 34, see also SEQ ID NOs: 40-44, 46, 47, 49-52; Bt,Bos taurus, SEQ ID NO: 35, see also SEQ ID NO: 38; Gg, Gallus gallus,SEQ ID NO: 36, see also SEQ ID NO: 39; Xl, Xenopus leavis, SEQ ID NO:37; Ec, Escherichia coli, SEQ ID NO: 45, see also SEQ ID NO: 48.

FIG. 2 shows structure of SoPIP2;1 (SEQ ID NO: 33) tetramer. SoPIP2;1(SEQ ID NO: 33) tetramer viewed from the extracellular side (a) and twoof the monomers viewed from the inside of the tetramer (b). The oxygensof water molecules and the Cd.sup.2+ ion are indicated as spheres.

FIG. 3 shows structural comparisons of eukaryotic aquaporins. Overlay ofAQP0, AQP1 and SoPIP2;1 (SEQ ID NO: 33). The D-loop with His193 and Leu197 (SEQ ID NO: 33) is blocking the pore in SoPIP2;1 whereas the D-loopsof AQP0 and AQP1 are occupying the same space as the C-terminal regionin SoPIP2;1 ending with Ser274 (SEQ ID NO: 33). The Cd.sup.2+ ion isindicated by the sphere at the lower left side of the figure.

FIG. 4 shows representation of the closed conformation of SoPIP2;1 (SEQID NO: 33). Representation of the closed conformation of SoPIP2;1 (SEQID NO: 33). a, The pore diameter of the closed conformation of SoPIP2;1(SEQ ID NO: 33) calculated using HOLE represented as a funnel with dotsillustrating the pore boundaries. b, A close up view of the pore nearthe gating region of loop D characterized by Leu197, Pro195 and Val194(SEQ ID NO: 33).

FIG. 5 shows electron density at the sites of regulation of SoPIP2;1(SEQ ID NO: 33) by phosphorylation and pH. Electron density at the sitesof regulation by phosphorylation and pH for SoPIP2;1 (SEQ ID NO: 33). a,Close up view of the divalent cation binding site showing the locationof the Cd²⁺ ion and the network of H-bonds linking Gly30 and Glu31 viaArg118 to Arg190 and Asp191 (SEQ ID NO: 33) of loop D. b, Close up viewof the phosphorylation residue Ser115 illustrating its H-bond to Glu31(SEQ ID NO: 33). c, Close up view of His193 (SEQ ID NO: 33). Whenprotonated an alternate conformation for His 193 may be adopted whichforms a salt bridge to Asp28 (SEQ ID NO: 33). d, Electron density forSer274 which contacts Pro199 and Leu200 (SEQ ID NO: 33) of a neighboringmonomer of the SoPIP2;1 (SEQ ID NO: 33) tetramer. All 2F^(obs)—F^(calc)maps are contoured at 1.0σ.

FIG. 6 shows difference anomalous density map for Cd²⁺ ion. Differenceanomalous density map illustrating the location of a single metal. a,Long distance view of the map. b, Close up view of the map near theassigned Cd²⁺ binding site. This map is contoured at 5σ.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In the context of the present application and invention, the followingdefinitions apply:

The term “pore diameter” is intended to mean the diameter at differentpositions within the pore of the aquaporin.

The term “closed conformation” refers to the structure of a closedaquaporin that do not permit the transfer of water molecules from oneside of the water channel to the other side due to a too small porediameter to allow water to pass

The term “aquaporin” is intended to mean a membrane channel protein thatfacilitate the flux of water and/or other small solutes acrossbiological membranes.

The term “gating mechanism” refers to the way the overall structure andthe positions of the individual amino acids change when the aquaporingoes from an open to a closed conformation or visa versa.

The term “ligand” is intended to mean any molecule (or part of amolecule) that is bound or is able to bind selectively andstoichiometrically to one or more specific sites on another molecule,for example a protein. Examples of ligands are peptides, smallmolecules, Cd²⁺, Ca²⁺, Mg²⁺, Mn²⁺ or any other divalent cation.

The synonymous terms “high resolution” and “atomic resolution” areintended to mean the minimum distance two atoms can be separated fromeach other and still be seen as two atoms and being below 2.5 Å.

The term “D-loop” is intended to mean at least a stretch of amino acidresidues being between the fourth and the fifth membrane spanning regionof an aquaporin, in SoPIP2;1 represented by amino acid residues 182-201(see, e.g., SEQ ID NO: 33), as shown in FIG. 1.

The term “homology modelling” is intended to mean a computational methodfor determining the structure of a protein based on its similarity toknown structures. Given the amino acid sequence of an unknown structureand the solved structure of a homologous protein, each amino acid in thesolved structure is mutated, computationally, into the correspondingamino acid from the unknown structure. The accuracy of structuresdetermined by homology modelling depends largely on the degree ofsimilarity between the unknown and the known protein sequence.

The term “atomic coordinates” or “structure coordinates” is intended tomean mathematical coordinates that describe the positions of atoms incrystals of the aquaporin.

The diffraction data obtained from the crystals are used to calculate anelectron density map of the repeating unit of the crystal. The electrondensity maps are used to establish the positions (i.e. coordinates X, Y,and Z) of the individual atoms within a single aquaporin. Those of skillin the art understand that a set of structure coordinates determined byX-ray crystallography is not without standard error. For the purpose ofthis invention, any set of structure coordinates for an aquaporin fromany source has a root mean square deviation of non-hydrogen atoms ofless than 0.75 .ANG. when superimposed on the non-hydrogen atompositions of the said atomic coordinates (Berman et al., 2000, NucleicAcids Research, 28, 235-242) set forth in Appendix 1. Other examples ofaquaporin structures are 1TM8, 1YMG, 1J4N, 1RC2 and 1FX8.

In the list of atomic coordinates set forth in Appendix 1 the term“atomic coordinate” refers to the measured position of an atom in thestructure in Protein Data Bank (PDB) format, including X, Y, Z and B foreach. The assembly of “atomic coordinate” also refers to “atomiccoordinates” or “structure coordinates”. The term “atom type” refers tothe element whose coordinates are measured. The first letter in thecolumn defines the element. The term “X,Y,Z” refers to thecrystallographically defined atomic position of the element measuredwith respect to the chosen crystallographic origin. The term “B” refersto a thermal factor that measures the mean variation of an atom'sposition with respect to its average position.

The term “molecular modelling” or “molecular structural technique” isintended to mean the use of computers to draw realistic models of whatmolecules look like and to make predictions about structure activityrelationships of ligands and other agents. The methods used in molecularmodelling range from molecular graphics to computational chemistry.

The term “molecular dynamics simulations” is intended to mean computersimulations of the dynamic properties of a molecule such asconformational changes using e.g. the Gromacs simulation suite or VMD(Ref. Berendsen H. J. C., van der Spoel, D. and van Drunen, R., CompPhys Commun 91, 43 (1995) and Humphrey, W., Dalke, A. & Schulten, K.VDM: visual molecular dynamics. J Mol Graph 14, 33-8 (1996).)

The terms “bind”, “binding”, “bond”, “bonded”, when used in reference tothe association of “binding agents” such as atoms, molecules, chemicalgroups or ligands is intended to mean any physical contact orassociation of two or more atoms, molecules, or chemical groups (e.g.,the binding of a ligand with a protein subunit refers to the physicalcontact between the ligand and the protein subunit). Such contacts andassociations include covalent and non-covalent types of interactions.

An Aquaporin

In a first aspect, the invention relates to an isolated aquaporin havinga bound ligand, wherein said ligand close the conformation of saidaquaporin and inhibit and/or reduce water transport of said aquaporin,and/or a high resolution structure of an isolated aquaporin in a closedconformation characterised by the coordinates set forth in Appendix 1.Examples of other aquaporins are those listed in FIG. 1, such as AQP0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 (see, e.g., SEQ ID NOs: 34-44, 46,and 47) or SoPIP2:1 (SEQ ID NO: 33). Said ligand may be a ligand asdefined above. Examples of ligands are Cd²⁺, Ca²⁺, Mg²⁺, Mn²⁺ or anyother divalent cation. The ligand may be bound at different sites on theaquaporin, such as at the cytoplasmic side. The ligand may for examplebe a non-covalently bound ligand. The ligand bound closed conformationcan be defined by a structure at high resolution. By determination ofthe structure of an isolated and crystallized aquaporin, a closedaquaporin conformation was obtained for the first time at atomicresolution. This enabled the possibility to deduce the gating mechanismof an aquaporin for the first time. The structure of such a closedaquaporin will enable the possibility to design new pharmaceutical(e.g., diuretics and inhibitors of angiogenesis) and cosmeceutical(e.g., antiperspirants) compounds that either stabilize the closedconformation or the open conformation. Since aquaporins are evolutionaryvery well conserved all the way from bacteria to plants and mammals,such compounds can be effective on aquaporins in many or all species.The atomic structure of a closed aquaporin such as SoPIP2;1 (SEQ ID NO:33) can also be used for structure based drug design and also fordesigning screening methods, both in silico and in vitro, foridentifying inhibitors and activators of aquaporins. By obtaining thestructure of a closed aquaporin and then using that particular structureit will be possible by genetic engineering to modify the gating andfunctioning of plant aquaporins, and to generate new plant varietieswith improved stress tolerance against drought.

Furthermore, the invention relates to an aquaporin having a porediameter of around 2.1 Å at the constriction region and less than thatin the pore towards the cytosolic vestibule when the aquaporin isclosed. The pore diameter is defined by the boundaries of the waterconducting pore. The pore diameter may be measured by the use of aprogram such as the HOLE program described by Smart et al., Biophys J65, 2455-2460 (1993). The structure was determined using a crystallisedform of an aquaporin wherein the aquaporin prior to crystallisation wasproduced in Pichia pastoris, i.e., overproduction of a heterologouseukaryotic protein as described below under the Examples. The structurewas solved at 2.1 Å resolution.

Additionally the aquaporin may be eukaryotic, such as selected from thegroup consisting of human, plant or animal aquaporins. Examples of plantspecies are spinach, sugar beet, Arabidopsis, maize, rice, wheat,barley, oats and mammalian species are human, bovine, sheep and othermammals, along with non-eukaryotic organisms such as yeast or bacteria.One specific example being the aquaporin from the plant spinach(SoPIP2;1, SEQ ID NO: 33), the present invention, was crystallised as atetramer displaying extended hydrophobic interactions between monomersas shown in FIG. 2. By the use of the X-ray crystal structure ofSoPIP2;1 (SEQ ID NO: 33) and then overlay the X-ray structure with thestructure of another aquaporins, such as the bovine AQP0 (16) (SEQ IDNO: 35) and AQP1(13) (SEQ ID NO: 38) it was possible to identify thatthey had an identical structural core consistent with the “hour-glassmodel” (19), differing only by 0.8 Å r.m.s.d. on Cα atoms within thetransmembrane regions (FIG. 3). SoPIP2;1 (SEQ ID NO: 33), bovine AQP0(16) (SEQ ID NO: 35) and AQP1 (13) (SEQ ID NO: 38). Likewise, thehalf-helices formed by loops B (cytosolic) and E (extracellular), withthe Asn-Pro-Ala aquaporin signature motif at the N terminal ends, foldinto the channel from opposite sides of the membrane and together createa seventh transmembrane region, which is perfectly preservedstructurally. Seven water molecules are observed within the SoPIP2;1(SEQ ID NO: 33) channel (FIG. 3), revealing an unbroken water networkstretching almost fully through the pore with a maximum distance of only3.1 Å between each water. The D-loop of the aquaporin or parts thereofbeing involved in the closed conformation of the aquaporin. The D-loopor part thereof being involved in the gating mechanism like a door whichopens and closes. A key residue in this respect is the fully conservedLeu197 (SEQ ID NO: 33) of loop D in SoPIP2;1, which inserts into acavity near the entrance of the channel and, in combination with His99,Val104 and Leu108 (FIG. 4 b, SEQ ID NO: 33), creates a hydrophobicbarrier blocking the pore. Calculations of the channel width, using HOLE(23) establish that the pore narrows to a diameter of approximately 1.4Å at Leu197 and narrows further to 0.8 A near Pro195 and Val194 (FIG. 4,SEQ ID NO: 33). This compares with the minimum pore diameter of 2.1 Åwithin the SoPIP2;1 (SEQ ID NO: 33) constriction region (9) and isinsufficient to allow the passage of water. Loop D and Leu197 (SEQ IDNO: 33) are also key to understanding the molecular mechanism of channelopening or closure in response to specific biochemical signals. In FIGS.4 b and c two water molecules separated by 6.4 Å are visible on eitherside of the hydrophobic barrier associated with Leu197, and one of theseforms H-bonds to His99 and the main-chain oxygen of Pro195 (SEQ ID NO:33). Should a conformational change in loop D concomitantly displaceLeu197, Pro195 and Val194 (SEQ ID NO: 33), then a pathway between thesetwo water molecules extending into the cytosol would open. Analogy withthe 2.1 Å pore-diameter of the constriction region implies that adisplacement of these loop D residues by as little as 1.3 Å could besufficient to open the channel. It has been postulated that divalentcations may play a role in aquaporin regulation (1,2,21) and inhibition(24,25). In FIGS. 2 and 3 a heavy-metal (FIG. 6 shows the anomalousdifference density map) is observed near loop D and is assigned as Cd²⁺since the addition of this ion improved the crystal quality. Cd²⁺ may bereplaced by another divalent cation in vivo, and a search for similarstructural motifs (26) revealed 13 PDB entries containing Ca². As such,we postulate that this metal binding site likely binds Ca² in vivo. Thissite is implicated in regulation since it serves to anchor loop D,through a network involving ionic interactions and H-bonds (FIG. 5 a),onto a short α-helix of the N-terminus (FIG. 3) and thus appears to becritical for defining the unique conformation of loop D observed forSoPIP2;1 (SEQ ID NO: 33). Specifically, Arg190 and Asp191 of loop D areconnected to the side-chain of Arg118 (strictly conserved in PIPs) andGly30 via a H-bond network containing three water molecules (SEQ ID NO:33). Arg118 in turn forms H-bonds to Glu31 (strictly conserved in PIPs)which ligates the Cd²⁺ ion (FIG. 5 a, SEQ ID NO: 33). Significantly, thehydroxyl group of the conserved phosphorylation site Ser115 also forms aH-bond to Glu31 (FIG. 5 b, SEQ ID NO: 33). It is therefore apparent thatthe covalent attachment of a phosphate group onto the hydroxyl oxygen ofSer115 would significantly perturb the conformation of Glu31, whichwould result in disrupting the crucial water mediated H-bond networkfrom Arg118 to Arg190 and Asp191 (FIG. 5 a, SEQ ID NO: 33). We suggestthat the disruption of this anchoring network would profoundly alter theconformation of loop D, and the resulting structural change would besufficient to displace Leu197, Pro195 and Val194 (SEQ ID NO: 33) andthereby unplug the entrance into the aquaporin channel from the cytosol.It is even possible that this displacement may result in loop D adoptinga conformation somewhat closer to that of AQP0 and AQP1 (FIG. 3).

Conversely, when Ser115 is phosphorylated and the water channel is open,the protonation of His193 (SEQ ID NO: 33) (strictly conserved) closesthe channel (4). A mechanism for pH-regulated PIP gating also emergesfrom the structure of SoPIP2;1 (SEQ ID NO: 33). In FIG. 5 c theconformation of His193 (SEQ ID NO: 33) is shown. At low pH where His193is protonated, a simple rotation of the histidine side-chain (FIG. 5 c)would enable it to form a salt bridge to Asp28 (SEQ ID NO: 33)(conserved in PIPs as either Asp or Glu). In this manner the H-bondmediated anchor for loop D onto the N-terminus (FIG. 5 a), which wesuggest is lost upon Ser115 (SEQ ID NO: 33) phosphorylation, would berecovered. As such the structure of Ser115 (SEQ ID NO: 33)phosphorylated SoPIP2;1 at low pH can be expected to be similar to thatreported here, with the cytosolic side of the aquaporin being capped byloop D and Leu197, Pro195 and Val194 (SEQ ID NO: 33) effectivelyblocking the water channel.

A structural framework for aquaporin regulation may also be proposedwhen considering the phosphorylation of Ser274 (SEQ ID NO: 33)(conserved in PIP2 homologues). In this case, however, Ser274 (SEQ IDNO: 33) is distant from the Cd²+ site. Instead, Ser274 (SEQ ID NO: 33)is located in the C-terminal region, of SoPIP2;1 which extends towardsthe four-fold axis of the tetramer and interacts with the main-chainnitrogen of Pro199 (SEQ ID NO: 33) of an adjacent monomer (FIGS. 2 b, 5d), which is the final residue of loop D. Should Ser274 (SEQ ID NO: 33)become phosphorylated then this interaction would be profoundlyaffected, and the creation of a cluster of four negative phosphatecharges in close proximity should induce a significant conformationalchange in the C-terminal region. An interaction between Ser274 (SEQ IDNO: 33) of one monomer with residues in loop D of another monomer in theaquaporin tetramer also suggests an orchestrated regulation of themonomers within homotetramers.

The closed conformation of SoPIP2;1 (SEQ ID NO: 33) was crucial fordiscerning a gating mechanism and the positions of specific amino acidsin the closed conformation, that previously biochemical and geneticexperiments had identified as important for gating, immediately suggesthow the closed structure can be stabilized and destabilized.

According to a second aspect, the invention relates to an aquaporincomprising an atomic structure characterised by the coordinates setforth in Appendix 1 and to phases computed from the coordinates of saidatomic structure. A person skilled in the art can easily by the use ofthe coordinates set forth in Appendix 1 in combination with one or moremolecular structural technique, develop new aquaporins or binding agentssuch as inhibitors to aquaporins as well as modify the inhibitors.

The disclosed isolated aquaporin crystal structure of said aquaporin aswell as the coordinates characterising said crystal may be used toscreen for binding agents, or ligands, that stabilise and/or destabilisethe closed conformation of said aquaporin or to create homology modelsof closed aquaporins. Other uses are to identify binding agents(ligands) or identification of at least one genetic modification capableof affecting the gating mechanism of said aquaporin, such as by an insilico technique. Another use is the development of genetically modifiedplants, such as agricultural plants.

Accordingly, water channels are functionally characterized byheterologous overexpression in Xenopus laevis oocytes. The method isdescribed in detail in references 1 and 2. The oocytes have lowintrinsic water permeability, allowing detection of any increase inwater permeability due to the expressed aquaporin. In this system AQP1,2, 3, 4, 5, 7, 8, 9 and 10 (see, e.g., SEQ ID NOs: 38-42 and 44) havehigh water permeabilities compared to AQP0, 6 and 11 (see, e.g., SEQ IDNOs: 34-37, 43, and 46) that are considered as poor water channels(Table 1; see also table 1 in Castle N A, Drug Discov. Today. 2005, 10,485-93). AQPs with a high water permeability are primary targets forbinding agents, ligands, that stabilize and/or destabilize a closedconformation since only a minor or no effect is expected by modulationof a poor or non-functional water channel.

TABLE 1 Water permeabilities for mammalian aquaporins and SoPIP2;1 (SEQID NO: 33) Water P_(f)-values permeability^(a) (μm/s)^(b) Reference^(c)AQP0 Low 13 ± 2 38 (see, e.g., SEQ ID NOs: 34-37) AQP1 High 190 ± 20 38(see, e.g., SEQ ID NOs: 38 and 39) AQP2 High 100 ± 10 38 (see, e.g., SEQID NO: 40) AQP3 High  80 ± 20 38 AQP4 High 290 ± 10 38 (see, e.g., SEQID NO: 41) AQP5 High 100 ± 10 38 (see, e.g., SEQ ID NO: 42) AQP6 Low 7.4 ± 0.7 39 (see, e.g., SEQ ID NO: 43) AQP7 High 150 40 AQP8 High 205± 12 41 (see, e.g., SEQ ID NO: 44) AQP9 High 289 ± 66 42 AQP10 High  8743 AQP11 Low 15 ± 6 44 (see, e.g., SEQ ID NO: 46) AQP12 NK^(c) NK (see,e.g., SEQ ID NO: 47) SoPIP2;1 (SEQ ID NO: 33) High 110 2 ^(a)Waterpermeabilites were categorized as either high or low based onP_(f)-values, using a cut off value of 50 μm/s. ^(b)Osmotic waterpermeability (P_(f)-values, average ± SD) were determined byheterologous overexpression in Xenopus laevis oocytes. 5 to 50 ng ofcRNA encoding an aquaporin were injected. After a preincubation to allowprotein expression, oocytes were transferred to hypotonic media and theswelling rates were recorded and used to calculate the P_(f)-values.^(c)NK, not known

The invention also relates to a method of how to produce said aquaporin.The method comprises the steps of providing said aquaporin, adding aligand to said aquaporin, producing crystals and obtaining an aquaporinhaving a ligand bound to the cytoplasmic side of said aquaporin, whereinsaid ligand close the conformation of said aquaporin and inhibit and/orreduce water transport through said aquaporin. The method beingdescribed in the examples.

Methods of Using the Isolated Aquaporin

The invented isolated aquaporin having a closed conformation, whereinsaid closed conformation is obtained by binding a ligand to saidaquaporin as defined above, said crystal as well as said atomicstructure may be used in several applications such as to screen forbinding agents (ligand) that stabilise and/or destabilise the closedconformation of said aquaporin or to create homology models of closedaquaporins based on the closed structure as defined by the coordinatesset forth in Appendix 1, to create molecular dynamics simulations ofsuch homology models of eukaryotic or prokaryotic aquaporins, toidentify binding agents or identification of at least one geneticmodification capable of affecting the gating mechanism of saidaquaporin, such as by an in silico technique or to develop geneticallymodified plants, such as agricultural plants.

According to another aspect, the invention relates to a method ofobtaining a binding agent (ligand) comprising: attaching a number ofaquaporins or parts thereof being defined above to a solid support andobtaining an array; adding a number of agents to said array; allowingsaid agents to bind to said aquaporins or parts thereof; removing saidagents which have not bound to said aquaporin or parts thereof andidentifying and obtaining said agents which have bound to saidaquaporins or parts thereof. In certain aspects one or more steps may beused such as attaching a number of aquaporins having a open conformationand then performing the same steps as with the aquaporins with a closedconformation. By adding such steps it is possible to discriminatebetween agents, which binds to the aquaporins having a closedconformation from those binding to the aquaporins having an openconformation. The most commonly used assay being a High ThroughputAssays. The power of high throughput screening is utilized to test newcompounds, which are identified or designed for their ability tointeract with an aquaporin of the invention. (For general information onhigh-throughput screening see, for example, Devlin, 1998, HighThroughput Screening, Marcel Dekker; U.S. Pat. No. 5,763,263). Highthroughput assays commonly use one or more different assay techniquesincluding, but not limited to, those described below. Said solid supportmay be any solid support such as a column, an array, a membrane, asandwich assay, competitive or competition assay, latex agglutinationassay, homogeneous assay, micro-titre plate format and themicro-particle based assay.

The aquaporin may be bound to the support by for instance covalentattachment, hydrophobic interactions and/or ionic interactions. Thenumber of agents may be a mixture of different agents, natural,synthetic, semisynthetic or a mixture thereof and organic compounds.After the agents have had the opportunity to bind to the aquaporin, theunbound agents are removed by for example washing by the use of a watersolution of buffers such as TRIS, PBS or MOPS. After removal of theunbound agents, the bound agents are to be identified which may beperformed by for example NMR, MS and antibodies, such as monoclonalantibodies. The antibodies can be labelled in various ways, including:enzyme-linked immunosorbent assay (ELISA); radioimmuno assay (RIA);fluorescent immunoassay (FIA); chemiluminescent immunoassay (CLIA); andlabeling the antibody with colloidal gold particles (immunogold).

According to a further aspect, the invention relates to a method ofobtaining an aquaporin binding agent comprising: using the atomiccoordinates as defined above and at least one molecular structuraltechnique to determine which agents interacts with an aquaporin andidentifying and obtaining said aquaporin binding agent. Such a methodmay also contain one or more additional steps, such as those mentionedabove in which open aquaporins are used in the same way as theaquaporins having a closed conformation to discriminate between agentswhich binds one or both of the different types of aquaporins. For basicinformation on molecular modelling, see, for example, M. Schlecht,Molecular Modelling on the PC, 1998, John Wiley & Sons; Gans et al.,Fundamental Principals of Molecular Modelling, 1996, Plenum Pub. Corp.;N.C. Cohen (editor), Guidebook on Molecular Modelling in Drug Design,1996, Academic Press; and W. B. Smith, Introduction to TheoreticalOrganic Chemistry and Molecular Modelling, 1996. The molecularstructural technique may be one of MOSFLM, SCALA, MOLREP, REFMAC5,NCSREF and CNS.

According to a further aspect, the invention relates to a method ofobtaining a modified agent comprising: using the atomic coordinates asdefined above and at least one molecular modelling technique todetermine how to modify the interaction of an agent with an aquaporin;modifying said agent based on the determination obtained in step (a) andproducing and obtaining a modified agent. The modifications of the agentmay be addition, elimination, modification or substitution of functionalgroups. There are several softwares that may be used and they include asfollows; GRID (Goodford, P. J., “A Computational Procedure forDetermining Energetically Favourable Binding Sites on BiologicallyImportant Macromolecules” J. Med. Chem., 28, pp. 849-857 (1985)).

The use of software such as GRID, a program that determines probableinteraction sites between probes with various functional groupcharacteristics and the macromolecular surface, is used to analyze thesurface sites to determine structures of similar inhibiting proteins orcompounds. The GRID calculations, with suitable inhibiting groups onmolecules (e.g., protonated primary amines) as the probe, are used toidentify potential hotspots around accessible positions at suitableenergy contour levels. GRID is available from Oxford University, Oxford,UK.

MCSS (Miranker, A. and M. Karplus, “Functionality Maps of Binding Sites:A Multiple Copy Simultaneous Search Method.” Proteins: Structure,Function and Genetics, 11, pp. 29-34 (1991)). MCSS is available fromMolecular Simulations, Burlington, Mass.

AUTODOCK (Goodsell, D. S. and A. J. Olsen, “Automated Docking ofSubstrates to Proteins by Simulated Annealing” Proteins: Structure.Function, and Genetics, 8, pp. 195-202 (1990)). AUTODOCK is availablefrom Scripps Research Institute, La Jolla, Calif.

DOCK (Kuntz, I. D. et al., “A Geometric Approach to Macromolecule-LigandInteractions” J. Mol. Biol., 161, pp. 269-288 (1982)).

The program DOCK may be used to analyze an active site or ligand bindingsite and suggest ligands with complementary steric properties. DOCK isavailable from University of California, San Francisco, Calif.

Once suitable chemical entities, compounds, or agents have beenselected, they can be assembled into a single ligand or compound orinhibitor or activator. Assembly may proceed by visual inspection of therelationship of the fragments to each other on the three-dimensionalimage displayed on a computer screen in relation to the atomiccoordinates of the aquaporin and/or its complexes with analogues. Thiswould be followed by manual model building using software such as Quantaor Sybyl.

Useful programs to aid one of skill in the art in connecting theindividual chemical entities, compounds, or agents include but are notlimited to:

CAVEAT (Bartlett, P. A. et al, “CAVEAT: A Program to Facilitate theStructure-Derived Design of Biologically Active Molecules”. In molecularRecognition in Chemical and Biological Problems”, Special Pub., RoyalChem. Soc., 78, pp. 82-196 (1989)).

Several methodologies for searching three-dimensional databases to testpharmacophore hypotheses and select compounds for screening areavailable. These include the program CAVEAT (Bacon et al. J. Mol. Biol.,225: 849-858 (1992)) which uses databases of cyclic compounds which canact as “spacers” to connect any number of chemical fragments alreadypositioned in the active site. This allows one skilled in the art toquickly generate hundreds of possible ways to connect the fragmentsalready known or suspected to be necessary for tight binding. CAVEAT isavailable from the University of California, Berkeley, Calif.

3D Database systems such as MACCS-3D (MDL Information Systems, SanLeandro, Calif.). This area is reviewed in Martin, Y. C., “3D DatabaseSearching in Drug Design”, J. Med. Chem., 35, pp. 2145-2154 (1992).

HOOK (available from Molecular Simulations, Burlington, Mass.).

The invented crystal of the aquaporin may be used for several purposesincluding the methods mentioned above to gain information, which can beused to identify compound as well as modify aquaporins. By obtaining thestructure of a closed aquaporin it will be possible to use the knowledgeof that particular structure to modify the gating. This can in plants bedone by direct genetic engineering of aquaporins in order to improvestress tolerance, e.g. against drought stress. In mammalian speciespharmaceutical compounds that stabilize the closed or the openconformation of aquaporins can be designed based on the closedconformation of SoPIP2;1 (SEQ ID NO: 33). Such inhibitors and activatorsare candidate pharmaceutical and cosmeceutical compounds, e.g.antiperspirants.

In a final aspect, the invention relates to the use of a ligand to closethe conformation of an aquaporin. The ligand to be used being definedand mentioned above.

Following examples are intended to illustrate, but not to limit, theinvention in any manner, shape, or form, either explicitly orimplicitly.

EXAMPLES Example 1 Expression in Pichia pastoris (as Described in Ref28)

The SoPIP2;1 (SEQ ID NO: 33) cDNA (GeneBank accession number L77969) wasoriginally amplified using the forward primer EcoRI-YPM28A(5′-CGGAATTCAAAATGTCTAAGGAAGTAAGT-3′, SEQ ID NO:1) and the reverseprimer PM28A-REV (5′-GAAGATCTTTAATTGGTAGGGTTGCT-3′, SEQ ID NO:2). Theforward primer has a EcoRI restriction site (underlined) and yeast startcodon. The reverse primer has the original stop codon after PM28A and aB gill restriction site (underlined). The PCR product was cloned intopPICZB (Invitrogen) and the resulting plasmid pPM28A-PICZ was sequenced.

PM28A constructs were transformed into the wild-type P. pastoris strainX-33 (Invitrogen) and transformants with the highest expressionaccording to immunostaining (TetraHis antibodies, Qiagen) were selectedand grown on a large scale.

Example 2 Purification of SoPIP2;1 (as Described in Ref 28 and 36)

The strain was grown in a 3 L fermentor typically resulting in 230 g wetcells/L culture after 24 h of methanol induction. Before breaking, thecells were resuspended in Breaking Buffer (50 mM potassium phosphate, pH7.5, 5% glycerol), frozen in liquid nitrogen and broken using anX-press. Unbroken cells were collected at 10 000 g, 30 min at 4° C. The10 000 g supernatant was further centrifuged at 100 000 g, 1.5 h at 4°C. to collect the membrane fraction. Peripheral membrane proteins andproteins adhering to the membranes were removed by urea (4 M urea, 5 mMTris-HCl, pH 9.5, 2 mM EDTA, 2 mM EGTA)/alkali (20 mM NaOH) treatment.The membrane was washed in each buffer and collected by centrifugationafter each wash as described above. SoPIP2;1 (SEQ ID NO: 33) wassolubilised in 5% OG (Anatrace) in Buffer A (20 mM HEPES-NaOH, pH 7.0,50 mM NaCl, 10% glycerol, 2 mM beta-mercapto ethanol) at roomtemperature for 30 min. Solubilised material was collected at 160 000 g,30 min at 4° C. Solubilised material was loaded on a Resource S column(20 mM HEPES-NaOH, pH 7.0, 1% OG) followed by a Superdex 200 column (20mM Tris-HCl, pH 7.5, 100 mM NaCl, 1% OG).

Example 3 Crystallisation

The purified sample was concentrated using a VivaSpin 20 concentrator(cutoff MW 10 kDa, VivaScience) to a final concentration of 15 mg/ml.

Crystals were obtained by the hanging drop vapour diffusion technique. 1μl of sample was mixed 1:1 with the reservoir solution containing 0.1 MTris-HCl pH 8.0, 30% PEG 400 and 0.1 M NaCl. 0.1 M CdCl₂ was added tothe drop in a 1:10 ratio.

The crystallisation setups were left to equilibrate at 4° C. Crystalsappeared within a few days and reached the maximum dimension of 0.1 μmwithin 1 week. Crystals were directly frozen in liquid nitrogen withoutthe need for further cryo-protecting.

X-ray diffraction data collection: A complete data set to 2.1 Åresolution was collected from a frozen crystal at −80° C. at the SwissLight Source (SLS) beamline X06SA, Switzerland. Image data wereprocessed using MOSFLM and scaled using SCALA of the CCP4 suite (29).Crystals belong to the space group I4 with 2 molecules in the asymmetricunit. The cell dimensions are a=b=90.0 Å, c=188.9 Å.

The atomic structure characterised by the coordinates are set forth inAppendix 1.

TABLE 1 Data collection and refinement statistics Data Collection Spacegroup I4 Cell dimensions a, b, c (Å) 90.0, 90.0, 188.9 α, β, γ(°) 90.0,90.0, 90.0 *Resolution (Å) 40.0-2.1 (2.27-2.1) *†R_(sym) 0.098 (0.431)*I/σI 5.0 (1.8) *Completeness (%) 99.8 (99.8) *Redundancy 3.9 (3.2)Refinement *Resolution (Å) 40.0-2.1 (2.27-2.1) No. reflections 41486‡R_(work)/§R_(free) 0.181/0.208 No. atoms Protein 3756 Ligand/ion 2Water 200 B-factors Protein 29.7 Ligand/ion 30.6 Water 39.6 R.m.sdeviations Bond lengths (Å) 0.02 Bond angles (°) 1.540 *Values inparentheses indicate statistics for the highest resolution shell.†R_(sym) = Ó|I_(o) − <I> I/Ó I_(o) × 100%, where I_(o) is the observedintensity of a reflection and <I> is the average intensity obtained frommultiple observations of symmetry related reflections. ‡R_(work) =Ó||F_(obs)| − k|F_(calc)||/Ó|F_(obs)| × 100%. §R_(free) is calculatedfrom 5% of the data which was excluded from refinement.

Example 4 Molecular Replacement & Structural Refinement

Molecular replacement was carried out using the program MOLREP from theCCCP4i-program suite with the coordinates of bovine AQP1 (PDB entry1J4N) (SEQ ID NO: 38) as the model. Using two copies of the model in themolecular replacement search, a clear solution with a correlationcoefficient and R-factor of 34.3% and 53.1% respectively was found. Thecrystal packing was checked using the program O(30) and there were nooverlaps between molecules. Automated model building was carried outusing ARP/WARP(31) and the resulting model was docked to the correctsequence using GUISIDE2. The calculated electron density map at thisstage was already of very good quality. Water molecules were pickedusing ARP/WARP. The model was subjected to multiple rounds of refinementin REFMAC5 (29), NCSREF (29) and CNS (32) with manual rebuilding in Obetween each round. During refinement, NCS restraints between the twomolecules in the asymmetric unit (molecule A and B) were used. Thecurrent model contains 251 residues (24-274) and one Cd²⁺ each formolecule A and B and 200 water molecules. The R-factor and Rfree are18.1% and 20.8% respectively. The quality of the structure was checkedin PROCHECK (33).

Assignment of Cd²⁺: A single metal binding site was unambiguouslyobserved as a dominant peak (still visible at 10.0σ) in the anomalousdifference density map (FIG. 6) and was identified as a divalent cationdue its coordination properties. Since the addition of 0.1 M CdCl₂ tocrystals improved diffraction from 3.7 Å to 2.1 Å resolution, we furtherassigned this cation as Cd²⁺. This assignment was justified byrecovering a B-factor of 29.8 Å² for the Cd²⁺ after refinement, similarto that of the side chains of its two protein ligands (average B-factorof 27 Å²). A search for similar binding sites using SPASM (34) yielded13 hits (PDB entries 1BQQ; 1CGE; 1CGL; 1HY7; 1JK3; 1M31; 1MMQ; 1MNC;1Q3A; 1RM8; 1RMZ; 1ROS; 830C) containing Ca²⁺ binding sites, and noother metals were found. We therefore make the identification that theCd²⁺ binding site is likely to be Ca²⁺ in vivo.

Example 5 Characterising the Channel

The package HOLE (23) was used to calculate the pore diameter forSoPIP2;1 (SEQ ID NO: 33), (FIG. 4). With this package a pore diameter of2.1 Å was recovered at the constriction region, apparently smaller thanthe effective diameter of water. This paradox is resolved byappreciating that HOLE returns an average diameter assuming a sphericalpore, which is an approximation.

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1. A method of creating a model of a closed aquaporin of unknown3-dimensional (3D) structure, the method comprising: replacing one ormore amino acids of a an atomic structure of a closed aquaporin of known3D structure, wherein said atomic structure of a closed aquaporin ofknown 3D structure comprises the atomic coordinates set forth inAppendix 1, with a corresponding amino acid of the closed aquaporin ofunknown 3D structure; and producing a model of the 3D structure of theclosed aquaporin of unknown 3D structure.
 2. The method according toclaim 1, wherein said closed aquaporin of unknown 3D structure is amammalian aquaporin.
 3. An in-silico method of creating a homology modelof a closed aquaporin of unknown structure, the method comprising:computationally mutating at least one of the amino acid residues in ahigh resolution structure of a spinach aquaporin denoted SoPIP2;1characterized by the atomic coordinates set forth in Appendix 1 into thecorresponding amino acid residue of said aquaporin of unknown structure;producing said homology model of said aquaporin of unknown structure;and optionally computationally refining said homology model.
 4. Themethod according to claim 3, wherein said closed aquaporin of unknownstructure is a mammalian aquaporin.
 5. An in-silico method of creating ahomology model of a closed aquaporin of unknown structure, the methodcomprising: computationally mutating at least one of the amino acidresidues in a high resolution structure of a crystalline, isolated,closed aquaporin of SEQ ID NO:33, wherein said aquaporin is a spinachaquaporin denoted SoPIP2;1 and comprises a bound cadmium ion; thecrystal being in space I4 and having unit cell dimensions a, b, c (Å)90.0, 90.0, 188.9 and α, β, γ (°) 90.0, 90.0, 90.0, into thecorresponding amino acid residue of said aquaporin of unknown structure;producing said homology model of said aquaporin of unknown structure;and optionally computationally refining said homology model.
 6. Themethod according to claim 4, wherein said closed aquaporin of SEQ IDNO:33 is a crystal of an aquaporin having a high resolution structurecharacterized by the atomic coordinates set forth in Appendix
 1. 7. Themethod according to claim 4, wherein said closed aquaporin of unknownstructure is a mammalian aquaporin.